HIRANUMA APPLICATION DATA

Watercontents

Karl Fischer Titrator

Data No. KF20

Apr. 13,2022

Drugs and Medicines – KF Volumetry
Japanese Pharmacopoeia – Suitability test

1. Abstract
Water contents of drugs and medicines could be determined by Karl Fischer volumetric titrator. In
volumetric titration, the titrant have a factor which means the ability to react with how many milligrams ofwater per 1 mL of titrant. Factor is pre-determined before sample measurement and water content of sample iscalculated from consumed titrant volume within sample measurement.
In the method of “Water determination (Karl Fischer method)” in Japanese Pharmacopoeia Eighteenth
Edition, the suitability test is described as a verification procedure when modifying the test procedures. In thisreport, a case example of suitability test is introduced with using glycerin as a simulated measurement sample.
Reference
1) Japanese Pharmacopoeia Eighteenth Edition

2. Apparatus and Reagents(1) Apparatus
Titrator：

Karl Fischer Volumetric titrator

Titration cell：Standard cell

Sampler：

Syringe：

AQUALYTE KF3 (HIRANUMA)

AQV-series(2) ReagentsKF Titrant

An alternative if it is difficult to prepare
HYDRANAL Composit 5 (Honeywell)：

HYDRANAL Methanol Dry (Honeywell)

Measurement sample：

Glycerin (Guaranteed reagent)Standard material：

AQUALYTE Water Standard 10 (Assay: 10.01 mg/g, HIRANUMA)

Titration solvent(3) Sample

An alternative if it is difficult to prepare
HYDRANAL Water Standard 10 (Honeywell)3. Procedure
3.1. Factor determination of KF titrant(1) Fill 50 mL of titration solvent into the titration cell as shown in Fig.3.1.
(2) Start blanking to attain stable background.
(3) Draw DI water into syringe. And then weigh it and record its read (Size 1 [g]).
(4) Inject a few drops of water from rubber septum of titration cell as shown in Fig.3.2. Approximate
amount of addition is 20-40 mg. (40-60 mg when using Composite 5 for KF titrant)(5) Start titration. Measurement parameter is shown in Table 4.1.
(6) Weigh the syringe again and record its read (Size 2 [g]). The difference of (Size 1-Size 2 [g]) is set assample size.
(7) Repeat the measurement 3 times and obtain an average value for factor of KF titrant.
* The above sampling procedure is an example for a simulated sample. It should be performed according
to the actual sample and the suitability test to be implemented.

Titration solvent

Fig.3.1 Preparation of the titration solvent

Note(1) Do not touch the tip of needle to titration
solvent.
(2) When injecting, set the tip of needle as
below figure.

(3) Before pulling out the needle, draw alittle air to prevent the sample fromadhering to the inside of rubber septum.

Fig.3.2 Injection of sample

3.2. Procedure for suitability test
(1) Fill 50 mL of titration solvent into the titration cell as shown in Fig.3.1.
(2) Start blanking to attain stable background.
(3) Wash the syringe with the sample of glycerin and draw it into syringe. Then, weigh the syringe andrecord its read (Size 1 [g]).
(4) Inject the sample from rubber septum of titration cell as shown in Fig.3.2. The injection amount isadjusted so that the detected water (mg) is within 5 to 30 mgH2O.
(5) Start titration. Measurement parameter is shown in Table 4.2.
(6) Weigh the syringe again and record its read (Size 2 [g]). The difference of (Size 1-Size 2 [g]) is set as
sample size.
(7) After measurement of the sample is finished, the device, setting parameters, and reagents are usedwithout change, and the water standard is measured subsequently. Wash the syringe with the water
standard and draw it into syringe. Then, weigh the syringe and record its read (Size 1 [g]).
(8) Inject the water standard from rubber septum of titration cell as shown in Fig.3.2. The injection
amount is adjusted so that the detected water (mg) is 50 to 100 % of that of the sample measurement.
Weigh the syringe again (Size 2 [g]). The difference of (Size 1-Size 2 [g]) is set as sample size.
(9) Repeat the measurement of the water standard 5 times.
* The above sampling procedure is an example for a simulated sample. It should be performed according
to the actual procedure of the suitability test to be implemented with actual sample.
3.3 Analysis of suitability test result(1) Prepare the value required for analysis from measurement result.
M: Determined water in the sample measurement result (mgH2O)⇒ Initial water content determined for sample of glycerin (mgH2O)

M1-X: Amount of added water in the water standard measurement (mgH2O),“X” indicates the number of water standard measurement
Added amount of water standard (g) × Water assay of water standard (mgH 2O/g)⇒ Amount of added water (mgH2O)

M2-X: Amount of found water in the water standard measurement result (mgH2O),“X” indicates the number of water standard measurement⇒Amount of found water (mgH2O)(2) Plot as follows using spreadsheet software.
x-axisy-axisM1-1
M + M 2-1
M1-1 + M 1-2
M + M 2-1 + M 2-2
M 1-1 + M 1-2 + M 1-3
M + M 2-1 + M 2-2 + M 2-3
M 1-1 + M 1-2 + M 1-3 + M 1-4
M + M 2-1 + M 2-2 + M 2-3 + M 2-4
M 1-1 + M 1-2 + M 1-3 + M 1-4 + M 1-5
M + M 2-1 + M 2-2 + M 2-3 + M 2-4 + M 2-5(3) Create a regression line for the plots and find the following values.
b : Slope of regression linea : y-axis interceptd : x-axis intercept(4) The percentage errors e1 (%) and e2 (%) are calculated from the following formulae.
e1 = {(a－M)／M} × 100
e2 = {(|d|－M)／M} × 100

(5) From the following formula, calculate the water recovery rate r (%) for each of the 5 measurements of
the water standard, and calculate the average water recovery rate R (%).
r (%) = (M2-X／M1-X) × 100

4. Parameters and results
Table 4.1 Parameters for factor measurement of KF titrant
ItemCal Mode
7:KF Factor(By Pure water)
Interval Time30 sec
Max Volume20 mLMin Feed Vol.
0.01 mL
S.Timer0 min
KF Buret No.
1Item
KF Speed(OUT)
KF Speed(IN)Back Ground
Sample Size Input
Blank Value
E.P Detection
Solvent
C.P Level
E.P Level
Auto IntervalAuto Input

24 mL/min
24 mL/minON
Every Time
0 mL
uA
S,O,CE
150 uA
200 uA0g
OFF

Table 4.2 Parameters for sample measurement
Item
Cal Mode
0:Sample weight (net)
Interval Time
30 secMax Volume
20 mL
Min Feed Vol.
0.01 mL
S.Timer0 min
KF Factor
3.3917 mg/mL
KF Buret No.
1Item
KF Speed(OUT)
KF Speed(IN)Back GroundSample Size Input
Blank Value
Unit ModeE.P Detection
Solvent
C.P Level
E.P Level
Auto Interval

24 mL/min
24 mL/minON
Every Time0 mL
AUTOuA
S,O,CE
150 uA
200 uA0g* The above parameter settings are examples for a simulated sample. They should be set according to theparameters for suitability test to be implemented with actual sample.

Table 4.3 Results of factor measurement of KF titrant
Meas. SampleTitrant
KF factor
Statistic result
No. size (g) volume (mL) (mg/mL)
Water
10.0269
7.92
3.3995 Avg. 3.3917 mg/mL
20.0297
8.81
3.3735 SD
0.0158 mg/mL
30.03219.44
3.4022 RSD
0.47 %

SampleFig.4.1 Measurement result of the sample

Fig.4.2 Example of measurement results of the water standard
Table 4.4 Values used for regression line of suitability test

SampleAdded water
Meas. Sample
M 1-X
No. size (g)(mgH2 O)

Sample1Waterstandard

123
4
5

7.14590.65880.70010.67280.66800.7204-6.595
7.008
6.735
6.687
7.211

Found water
M 2-X(mgH2 O)

8.662
6.566
6.994
6.651
6.624
7.136

x-axisCumulative
added water(mgH2 O)-y-axisM + Cumulative
found water(mgH2 O)
-6.59513.60320.33727.02434.23515.22822.22228.87335.49742.633

Fig.4.3 Result of calculated regression line

Percentage
recoveryr (%)-99.57
99.80
98.76
99.06
98.96

Table 4.5 Result of suitability test for Japanese Pharmacopoeia

Item
Found water of sample M (mgH2O)
Average percent recovery R (%)
y-axis intercept a (mgH2O)
Slope of regression line b
x-axis intercept d (mgH2O)
Percentage error |e 1| (%)
Percentage error |e 2| (%)

Result
8.662
99.23
8.715
0.991
-8.7940.61
1.52

Standard
5～30 mgH2O
97.5～102.5 %0.975～1.025
2.5 % or less
2.5 % or lessCompliantOKOKOKOKOK

5. NoteAccording to the Japanese Pharmacopoeia Eighteenth Edition, the suitability test is implemented for theverification when the procedures of sample measurement are changed. Please check the latest version ofJapanese Pharmacopoeia before conducting the suitability test.
The condition parameters set in the device and the procedure of sample addition used in this report areexamples for simulated samples. Adapt these to the actual sample to be verified by the suitability test.
Even if the sample, parameters, and sample addition method are different, the analysis procedure ofsuitability test can be performed in the same way.
In addition, from the Eighteenth Edition of Japanese Pharmacopoeia, the suitability test was adopted andthe specific description on the preparation of Karl Fischer reagent was deleted. The purpose of this is asfollows; as premises for complying the suitability test, it is possible to select and use the appropriate KFreagent from commercially available products, considering the solubility and interfering reaction
according to the properties of the measurement sample.
Keywords：Karl Fischer, Volumetric titration, Japanese Pharmacopeia, Suitability test